Site-specific cleavage of nucleic acids by photoreactive conjugates

ABSTRACT

A process of forming a double strand cleavage in DNA includes providing a reaction mixture containing double stranded DNA having a break in a first strand defining a target site in a second strand. The method continues by adding to the reaction mixture a photoreactive lysine conjugate selected from a lysine-enediyne conjugate, a lysine-acetylene conjugate or a combination thereof, for a time sufficient for the lysine conjugate to bind to the DNA adjacent the target site. The reaction mixture is then irradiated with electromagnetic radiation sufficient to photoactivate the lysine conjugate to cleave the second strand adjacent the target site.

RELATED APPLICATION

This application claims priority from co-pending provisional applicationSer. No. 60/753,156, which was filed on Dec. 22, 2005, and which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to biochemical methods and, more specifically, toa method for achieving efficient cleavage of double stranded DNA orsite-selective cleavage of single stranded nucleotides.

BACKGROUND OF THE INVENTION

Restriction enzyme and siRNA technologies exist for site-specificknockout of genes. These two technologies are limited to one kind oftargeted molecule. The presently described system is more flexible andprovides an alternative method for directing the site-specificmodification of nucleic acids, particularly DNA.

Aside from the common double helix, DNA forms a wide range of structuralmotifs, such as hairpin loops, triplex, tetraplex, bulged structures, aswell as nicks and gaps. The individual structural features of thesemotifs make them potential candidates for specific targeting. Amongthese structural elements, nicks and gaps are promising for thedevelopment of sequence-specific DNA cleavage, since they alreadyfeature a break in the phosphate backbone of DNA.

Cleavage of the phosphate backbone of DNA can be caused by chemicalreagents such as radicals and by radiation damage. In order to survive,cells developed enzymatic mechanisms for the repair which workefficiently on single strand (ss) damage. Any further cleavage on theopposite strand at the damage site leads to double stranded (ds)cleavage, which is hard to repair. Ds cleavage requires either abifunctional reagent or detection and targeting of the damaged site. Theonly literature example of the latter is a complex natural antibiotic,bleomycin.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention was directed todeveloping a general method for artificial and sequence-specific nucleicacid modification, based upon, but not limited to, our system. Thisternary system is capable of targeting of nucleic acids. It consists ofthree elements:

-   -   a DNA-sequence, providing recognition of the nucleic acid        target, sequence selectivity;    -   a terminal phosphate group on DNA-sequence, serving as a        directing group for the ligand; and    -   a phosphate detecting ligand (e.g., Lys, Arg, Ala, etc.)-warhead        (e.g., enediyne, acetylene and other photoreactives) conjugate.

Our initial research efforts focused on the natural selectivity oflysine-enediyne conjugates towards a radioactively-labelled DNAoligomer. After irradiation in presence of lysine-enediyne conjugate andsubsequent treatment with piperidine, we observed cleavage that ismostly localized at the Guanosine (G)-sites of the labelled DNA-strand.It is a known that oxidative damage (such as this case) is localized toG-sites, due to the lower oxidation potential of G compared to othernucleobases. Moreover, damage inflicted on sites other than G will tendto migrate (“hole hopping”) to G over a distance of several nucleobases.A DNA-construct: 54mer with internal label near the 3′ end, full-lengthcounterstrand, is shown in FIG. 1.

These findings are summarized in the provisional application which isthe parent to the present application and have been submitted forpublication in a paper titled Internally Labelled Oligonucleotides:Investigation of Sequence Selectivity of DNA Photocleavage by Enediyne-,Fulvene-, and Acetylene-Lysine Conjugates, whose authors are BorisBreiner, Jörg C. Schlatterer, Serguei V. Kovalenko, Nancy L. Greenbaum,Igor V. Alabugin.

In a subsequent set of experiments, we tried to amplify the naturalpreference for cleavage at the G-sites; in particular, we placedphosphate groups on short, complementary counterstrands opposite to aGGG triad. Cleavage in these systems was clearly enhanced at the siteopposite to the phosphate groups (the Gs in bold, as shown in FIG. 2).Cleavage at the other G-sites was not affected, i.e., the cleavage atthe other G sites was as strong as it was in the experiments with acomplete counterstrand.

As shown in FIG. 2, the DNA-constructs from left to right are asfollows:

-   -   a DNA oligomer with short counterstrand bearing a 5′-terminal        phosphate group;    -   a DNA oligomer with short counterstrand bearing a 5′-terminal        phosphate group and a second counterstrand without a terminal        phosphate group; all bases are base-paired; and    -   a DNA oligomer with short counterstrand bearing a 5′-terminal        phosphate group and a second counterstrand bearing a        3′-phosphate group; the second strand is shortened to leave a        gap of 1 base between the two counterstrands.

In another set of experiments, the location of the phosphate group wasmoved to a site that was in-between two known G-cleavage sites. In thesecases, cleavage was amplified at the two cleavage sites that wereclosest to the location of the phosphate(s) on the counterstrand(s).

As shown in FIG. 3, DNA-constructs from left to right are as follows:

-   -   a DNA oligomer with short counterstrand bearing a 5′-terminal        phosphate group;    -   a DNA oligomer with short counterstrand bearing a 5′-terminal        phosphate group and a second counterstrand without a terminal        phosphate group; all bases are base-paired; and    -   a DNA oligomer with short counterstrand bearing a 5′-terminal        phosphate group and a second counterstrand bearing a        3′-phosphate group, the second strand is shortened to leave a        gap of 1 base between the two counterstrands.

A conclusion drawn from these experiments is that it appears the ligandbinds preferentially to the sites of the phosphate groups. The oxidativedamage migrates (in part) away from this site, but the damage is clearlymost prominent at the desired location.

The present invention, then, in general, includes the use of randomligand-warhead conjugates for site-specific damage of nucleic acids bycomplexation of phosphorylated, complementary RNA or DNA molecules. Thismethod constitutes a new generation of site specific effects bybioactive compounds, an advantage being that all that is required tomodify a known sequence of RNA or DNA is a short, complementary,phosphorylated DNA oligomer and a universal ligand attached to an activecompound.

Therefore, the present invention advantageously provides thatphotoreactive conjugates, for example, lysine conjugates, can identifyss damage sites with high selectivity and induce DNA cleavage at thestrand opposite to the damage site (as illustrated in Scheme 1). InScheme 1, two components are potentially responsible for damage siterecognition and subsequent cleavage: 1) formation of a hydrophobicpocket and 2) electrostatic interaction between the additional negativecharges due to the presence of terminal phosphate moieties at thenegatively charged DNA backbone ((−) signs) and the protonated amines oflysine moiety ((+) charges).

Use of this recognition for site-specific cleavage of single-strandednucleotides can be achieved using the following sequence of steps. A.Annealing process that positions recognition site opposite the target atthe original single strand oligonucleotide. B. Recognition of the targetsite by lysine conjugates. C. Sequence-selective photochemicalconversion of single stranded DNA cleavage into double strandedcleavage.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1 is a diagram of a DNA-construct: 54mer with internal label nearthe 3′ end, full-length counterstrand;

FIG. 2 shows DNA-constructs (from left to right): a DNA oligomer withshort counterstrand bearing a 5′-terminal phosphate group; a DNAoligomer with short counterstrand bearing a 5′-terminal phosphate groupand a second counterstrand without a terminal phosphate group (all basesare base-paired); and a DNA oligomer with short counterstrand bearing a5′-terminal phosphate group and a second counterstrand bearing a3′-phosphate group, wherein the second strand is shortened to leave agap of 1 base between the two counterstrands;

FIG. 3 shows DNA-constructs (from left to right): a DNA oligomer withshort counterstrand bearing a 5′-terminal phosphate group; a DNAoligomer with short counterstrand bearing a 5′-terminal phosphate groupand a second counterstrand without a terminal phosphate (all bases arebase-paired); and a DNA oligomer with short counterstrand bearing a5′-terminal phosphate group and a second counterstrand bearing a3′-phosphate group, the second strand being shortened to leave a gap of1 base between the two counterstrands;

FIG. 4 provides the structures and formulas for two lysine conjugatesreferred to as formula 1 and formula 2; both shown as unprotonatedamines.

FIG. 5 depicts the method of synthesizing lysine-enediyne conjugate 1;

FIG. 6 illustrates the method of synthesizing the lysine-acetyleneconjugate 2;

FIG. 7 shows the design of control DNA duplex 54mer and single strand(ss) damage sites, according to an embodiment of the present invention;

FIG. 8 diagrams a hypothetical mechanism for the present inventivemethod, showing a diagrammatic view of Scheme 1, where two componentsare potentially responsible for damage site recognition and subsequentcleavage: 1) formation of a hydrophobic pocket and 2) electrostaticinteraction between the additional negative charges due to the presenceof terminal phosphate moieties at the negatively charged DNA backbone((−) signs) and the protonated amines of lysine moiety ((+) charges). A.Annealing process that positions recognition site opposite the target atthe original single strand oligonucleotide. B. Recognition of the targetsite by lysine conjugates. C. Sequence-selective photochemicalconversion of single stranded DNA cleavage into double strandedcleavage.

FIG. 9 is a comparison between the intact DNA duplex A and constructs B,C, D, and E; the constructs (0.2 μM) were irradiated in presence ofcompound 1 (10 μM) in borate buffer (pH 7.6); top: autoradiogram of PAGEanalysis; the arrows show the position of the target site; bottom:histograms showing the quantified cleavage data, normalized relative tothe largest peak in construct A;

FIG. 10 illustrates a comparison between DNA construct A and constructsF, F′, G, and G′; the constructs (1 μM) were irradiated in presence ofcompound 1 (10 μM) in borate buffer (pH 7.6); the arrows show theposition of the target site; top: autoradiogram of PAGE analysis;bottom: histograms showing the quantified cleavage data, normalizedrelative to the largest peak in construct A;

FIG. 11 displays histograms showing the amount of frank cleavage at theG₂₄G₂₅G₂₆ triad in various constructs under identical conditions;intensities are relative to the largest peak in construct A. E and E′correspond to gapped constructs with and without terminal phosphategroups;

FIG. 12 is a comparison between DNA construct A and constructs C′, E′,C, and E; the constructs (1 M) were irradiated in presence of compound 1(10 M) in borate buffer (pH 7.6); top: autoradiogram of PAGE analysis,the arrows show the position of the target site.; bottom: histogramsshowing quantified cleavage data, normalized relative to the largestpeak in construct A;

FIG. 13 shows a comparison between DNA constructs A and E in differentbuffer conditions; the constructs (1 M) were irradiated in presence ofcompound 1 (10 M) in left: borate buffer (pH 7.6, 20 mM); middle: boratebuffer (pH 7.6, 4 mM); right: phosphate buffer (pH 7.6, 20 mM); top:autoradiogram of PAGE analysis, the arrows show the position of thetarget site; bottom: histograms showing the quantified cleavage data,normalized relative to the largest peak in construct A; and

FIG. 14 is a comparison between DNA constructs A and E in differentbuffer conditions; the constructs (1 M) were irradiated in presence ofcompound 2 (10 M) in phosphate buffer; left: pH 6; middle: pH 7; right:pH 8; top: autoradiogram of PAGE analysis; the arrows show the positionof the target site; bottom: histograms showing the quantified cleavagedata, normalized relative to the largest peak in construct A.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Anypublications, patent applications, patents, or other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may, however, be embodied in many differentforms and should not be construed as limited to the illustratedembodiments set forth herein. Rather, these illustrated embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

Experimental Materials and Methods

The following description provides detailed instructions for carryingout the presently described invention and, particularly, all theexperimental procedures employed.

General information: All reagents used were purchased from Sigma andAcros Organics if not otherwise noted. All buffers were prepared andpH-adjusted at room temperature (25° C.).

Irradiation conditions: Samples were placed on ice at a distance of 20cm from 200 W Hg—Xe lamp (Spectra-Physics, Laser & Photonics OrielInstruments with long pass filter with 324 nm cut-on wavelength).

Lysine conjugates according to formulas 1 and 2 are shown in FIG. 4. Thesynthesis of compound 1 is described by Kovalenko, S. V. and I. V.Alabugin, Chem. Comm. 2005, 1444, a scientific paper which is accessibleto the skilled and which is incorporated herein by reference in itsentirety.

Synthesis of lysine-acetylene conjugate 1: as shown in FIG. 53,4-Bis(phenylethynyl)nitrobenzene

A mixture of 3,4-dibromonitrobenzene (4.2 g, 15 mmol),tris(triphenylphosphine)palladium chloride (0.5 g), and copper (I)iodide (0.15 g) in 70 ml of triethylamine was degassed byfreeze/pump/thaw technique (three times). Phenylacetylene (3.4 g, 34mmol) was added and the mixture was stirred at room temperature undernitrogen for 3 days. The amine was removed by rotary evaporation, andthen the residue was dissolved in CH₂Cl₂ and washed with water. Theorganic layer was dried (Na₂SO₄), filtered, and rotary evaporated.Chromatography (EtOAc/hexanes, 1:30) gave enediyne (3.7 g, 76%) asyellow solid: mp. 97-98° C. ¹H NMR (300 MHz, CDCl₃) δ 8.42 (d, J=2.4 Hz,1H), 8.16-8.12 (dd, J=2.4 Hz, J=8.4 Hz, 1H), 7.68 (d, J=8.4 Hz, 1H),7.61-7.59 (m, 4H), 7.42-7.35 (m, 6H). ¹³C NMR (68 MHz, CDCl₃) δ 146.6,132.4, 132.0, 131.9, 131.8, 129.4, 129.2, 128.6, 127.2, 126.7, 126.6,122.6, 122.3, 112.2, 98.8, 95.9, 87.0, 86.3. m/z (high res. El) 323.0947(Calc. C₂₂H₁₃NO₂ 323.0946).

3,4-Bis(phenylethynyl)phenylamine

A solution of 3,4-bis(phenylethynyl)nitrobenzene (2.5 g, 7.7 mmol),SnCl₂ (7.3 g, 39 mmol, 5 eq.), and HCl (5 mL, 10 eq.) in THF (20 mL) wasstirred at room temperature for 5 h. After neutralization with NaOH(1.0N solution), product was extracted with dichloromethane. Solvent wasevaporated and the residue was purified by column chromatography onsilica gel using chloroform as the eluent to yield 1.7 g (75%) of3,4-bis(phenylethynyl)phenylamine as a brown oil. ¹H NMR (300 MHz,CDCl₃) δ 7.67-7.61 (m, 4H), 7.42-7.35 (m, 7H), 6.85 (d, J=2.4 Hz, 1H),6.62-6.58 (dd, J=2.4 Hz, J=8.4 Hz, 1H), 3.85 (broad, 1H). ¹³C NMR (68MHz, CDCl₃) δ 146.3, 132.9, 131.5, 131.2, 131.8, 128.3, 128.2, 127.7,126.5, 123.7, 123.1, 117.3, 117.2, 115.1, 114.9, 92.8, 91.2, 88.9, 88.5.m/z (high res. ESI) 294.1283 (Calc. C₂₂H₁₆N 294.1283).

2,6-Diamino-hexanoic acid[3,4-bis(2,3,5,6-tetrafluoro-pyridin-4-ylethynyl)-phenyl]-amideDichloride

Boc₂Lys(OH) (1.5 g, 5.1 mmol) and aniline (1.77 g, 5.1 mmol) weredissolved in pyridine (20 mL). The solution was cooled to −20° C. andphosphorus oxychloride (0.95 ml, 10.2 mmol) was added dropwise withvigorous stirring. The reaction mixture was stirred for 1 h at −20° C.and then at room temperature for 3 h. The reaction mixture was quenchedwith ice/water and anilide was extracted with CH₂Cl₂. The organic phasewas washed with aqueous sodium bicarbonate, and saturated aqueous sodiumchloride. The organic phase was dried with Na₂SO₄ and the solvent wasevaporated in vacuo. The crude product was subjected to chromatographywith CH₂Cl/CH₃CN (10:1) as eluent and desired compound obtained as whitesolid (1.5 g, 47%): mp. 105-107° C. ¹H NMR (300 MHz, CDCl₃) δ 9.16(broad, 1H), 7.73 (s, 1H), 7.34-7.27 (m, 6H), 7.14-7.12 (m, 6H), 5.51(m, 1H), 4.64 (broad, 1H), 4.2 (broad, 1H) 2.9 (m, 2H), 2.4 (s, 2H),1.75 (m, 1H), 1.6 (m, 1H), 1.3 (20H). 13C NMR (68 MHz, CDCl3) δ 171.3,156.3, 137.7, 132.2, 131.6, 131.5, 128.2, 128.1, 126.3, 123.3, 122.3,122.2, 121.2, 119.1, 93.4, 92.8, 88.2, 88.1, 80.3, 79.1, 55.1, 39.6,31.8, 29.5, 28.4, 22.6. m/z (high res. ESI) 644.3094 (Calc. C₃₈H₄₃N₃O₅Na644.3100).

Boc-Protected compound (1 g) was dissolved in THF, and gaseous HCl waspassed through that solution for 2 h. Then solvent was evaporated, andsolid was washed with acetonitrile. After drying under vacuum thedesired compound was obtained as yellow solid (0.47 g, 70%): mp.295-297° C. (decomp.). 1H NMR (300 MHz, DMSO-d6) δ 8.4 (broad, 2H), 8.04(s, 1H), 8.03 (broad, 1H) 7.78-7.76 (d, J=8.4 Hz, 1H), 7.65-7.62 (d,J=8.4 Hz, 1H), 7.60-7.40 (m, 10H), 4.14 (broad, 1H) 2.77 (m, 2H), 1.88(m, 2H), 1.61 (m, 2H), 1.45 (m, 2H). 13C NMR (68 MHz, CDCl3+CD3OD) δ166.9, 136.9, 131.8, 131.1, 130.9, 128.1, 127.9, 125.9, 123.3, 122.6,122.4, 122.1, 121.4, 119.2, 93.2, 92.7, 87.4, 87.3 53.1, 38.9, 30.5,26.0, 28.4, 21.3. m/z (high res. ESI) 422.2223 (Calc. C₂₈H₂₈N₃O422.2232).

Synthesis of lysine-acetylene conjugate 2: as shown in FIG. 6

4-(Trimethylsilylethynyl)nitrobenzene. A mixture of p-bromonitrobenzene(2.02 g, 10 mmol), bis(triphenylphosphine)palladium(II) chloride (0.2 g,0.3 mmol), and copper(1) iodide (0.05 g, 0.3 mmol) in 40 ml ofN,N-diisopropylamine was degassed by freeze/pump/thaw technique (threetimes). Trimethylsilylacetylene (1.2 g, 12 mmol) was added and themixture was refluxed for 12 hours. After removal of amine by rotaryevaporation, the residue was dissolved in CH₂Cl₂ and washed with water.The organic layer was dried (Na₂SO₄), filtered, and rotary evaporated.The residue was purified by column chromatography (EtOAc/hexanes, 1:10)to afford 4-(trimethylsilylethynyl)nitrobenzene (1.53 g, 70%).(Procedure adapted from Takahashi, S; Kuroyama, Y; Sonogashira, K;Hagihara, N. Synthesis, 1980, 627.)

4-(2,3,5,6-Tetrafluoropyridin-4-ylethynyl)nitrobenzene. A solution of4-(2,3,5,6-trimethylsilylethynyl)nitrobenzene (0.95 g, 4.3 mmol) in DMF(10 mL) was added to the mixture of pentafluoropyridine (0.73 g, 4.3mmol) and CsF (1.0 g, 6.5 mmol, 1.5 eq.) in DMF (10 mL) for 4 h usingsyringe pump. The reaction mixture was stirred constantly during theaddition. Water (20 mL) and dichloromethane (50 mL) were added after theaddition was complete. Organic phase was separated, washed with ammoniumchloride aqueous solution. Solvent was evaporated. The residue waschromatographed (EtOAc/hexanes, 1:15) to provide4-(2,3,5,6-tetrafluoropyridin-4-ylethynyl)nitrobenzene as a slightlyyellow solid (0.77 g, 60%): mp. 155-158° C.; ¹H NMR (300 MHz, CDCl₃) δ8.32-8.29 (d, J=9 Hz, 2H), 7.82-7.79 (d, J=9 Hz, 2H). ¹³C NMR δ (68 MHz,CDCl₃) 148.5, 145 (m, 143 (m, 141 (m, 140 (m, 133.2, 126.9, 123.9, 116(m), 103.1. ¹⁹F (282 MHz, CDCl₃) δ −137.5 (m, −89.6 (m.

4-(2,3,5,6-Tetrafluoropyridin-4-ylethynyl)aniline. A solution of SnCl₂(1.6 g, 8.4 mmol) in THF (5 ml) was slowly (1.5 hours) added to themixture of 4-(2,3,5,6-tetrafluoropyridynylethynyl)nitrobenzene (0.5 g,1.7 mmol) and HCl (1 ml) in THF (5 ml). The reaction mixture was stirredat the room temperature for 2 hours. After neutralization with NaOH (1.0N solution), product was extracted with dichloromethane. Solvent wasevaporated and the residue was purified by column chromatography onsilica gel using chloroform as the eluent to yield3,4-bis(tetrafluoropyridinylethynyl)aniline as a yellow solid (0.39 g,86%): mp. 164-165° C.; ¹H NMR (300 MHz, CDCl₃) δ 7.44-7.42 (d, J=8.7 Hz,2H), 6.68-6.65 (d, J=8.7 Hz, 2H), 4.04 (broad, 2H). ¹³C NMR δ (68 MHz,CD₃CN) 152.0, 146 (m, 144 (m, 142 (m, 140 (m, 135.0, 115.1, 110 (m,107.9, 73 (m. ¹⁹F (282 MHz, CDCl₃) δ −136.5 (m, −88.6 (m.

/2,6-Diamino-hexanoic acid[4-(2,3,5,6-tetrafluoro-pyridin-4-ylethynyl)-phenyl]-amide Dichloride.Boc₂Lys(OH) (1.3 g, 3.7 mmol) and aniline (0.34 g, 3.7 mmol) weredissolved in pyridine (10 mL). The solution was cooled to −20° C. andphosphorus oxychloride (0.5 ml, 5.2 mmol) was added dropwise withvigorous stirring. The reaction mixture was stirred for 1 h at −20° C.and then at room temperature for 10 h. The reaction mixture was quenchedwith ice/water and anilide was extracted with CH₂Cl₂. The organic phasewas washed with aqueous sodium bicarbonate and saturated aqueous sodiumchloride. The organic phase was dried with Na₂SO₄ and the solvent wasevaporated in vacuo. The crude product was subjected to chromatographywith CH₂Cl/CH₃CN (10:1) as eluent and desired compound obtained as whitesolid (1.1 g, 51%): mp. 137-139° C. ¹H NMR (300 MHz, CDCl₃) δ 9.15(broad, 1H), 7.58-7.55 (d, J=8.4 Hz, 2H), 7.49-7.46 (d, J=8.4 Hz, 2H),5.42 (broad, 1H) 4.7 (broad, 1H), 4.26 (broad, 1H), 3.12 (broad, 2H),1.9 (m, 1H), 1.7 (m, 1H), 1.5 (m, 2H), 1.4 (s, 18H). ¹³C NMR δ (68 MHz,CDCl₃) 171.2, 156.5, 156.3, 145 (m, 143 (m, 142 (m, 140.2, 140 (m,133.2, 119.3, 117 (m, 115.5, 106.8, 80.7, 79.38, 73 (m, 55.2, 39.6,31.3, 29.6, 28.4, 28.3, 22.5. ¹⁹F (282 MHz, CDCl3) δ −139.0 (m, −91.0(m. 0.6 g of this compound was dissolved in THF, and gaseous HCl waspassed through that solution for 1 h. The solvent was evaporated, andsolid (0.18 g, 40%) was recrystallized from ethanol. Mp. 260-264°(decomp.). ¹H NMR (300 MHz, DMSO) δ 8.35 (broad, 1H), 7.8-7.82 (d, J=9Hz, 2H), 7.78 (broad, 1H) 7.72-7.69 (d, J=9 Hz, 2H), 4.04 (broad, 1H),2.76 (broad, 2H), 1.84 (m, 2H), 1.6 (m, 2H), 1.4 (m, 2H). ¹³C NMR δ (68MHz, CDCl₃+CD₃OD) 165.9, 144 (m, 143 (m), 141 (m, 140 (m, 139.4, 132.6,119.2, 115.7, 105.9, 73 (m, 52.9, 38.4, 30.3, 26.0, 21.1. ¹⁹F (282 MHz,CDCl₃) δ −140.0 (m, −92.0 (m.

Nucleic Acids

We used an internally ³²P-labeled 54 nt DNA (BW 54s, 5′-TAA TAC GAC TCACTA TAG GCC CAG GGA AAA CTT GTA MG GTC TAC CTA TCT*ATT, a ³²P labelindicated by asterisk; and identified herein as SEQ ID No:1). Thissequence incorporates several isolated guanine (G) nucleotides as wellas GG diads, GGG triads, and an AT tract, the latter serving as anatural binding site for a family of lysine-conjugates. We havepreviously shown that a combination of AT-selectivity of binding andG-selectivity of activation through photoinduced electron transfer (PET)can be used for selective targeting of guanines flanking the AT-tract.See: Breiner, B. and J. C. Schlatterer, S. V. Kovalenko, Nancy L.Greenbaum and I. V. Alabugin in Angew. Chem. Int. Ed. 2006, 45, 3666, ascientific paper accessible to the skilled and which is incorporatedherein by reference in its entirety.

³²P-labelling of DNA oligomers for phototriggered damage: The synthesisof BW 54 s was described previously. Id. Aqueous 2 μM dsDNA solution wasgenerated by mixing the appropriate DNA oligomers (see below),incubation at 94° C. for 3 minutes and cooling down to room temperaturewithin 60 minutes. Typically ˜1000 cpm were used per experiment. A+G andC+T sequence markers were produced according to the Maxam-Gilbertsequencing protocol (Sambrook, J., E. F. Fritsch and T. Maniatis, 1989,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press,Plainview, N.Y.; a learned treatise known to the skilled andincorporated herein by reference in its entirety) and were loaded on thegels in the lanes labelled A+G and C+T, respectively.

The following oligomers were annealed with BW 54s to generate constructsA-G′, which are set forth below:

A:

BW 54 as (SEQ ID NO:2): 5′-AAT AGA TAG GTA GAC CTT TAC AAG TTT TCC CTGGGC CTA TAG TGA GTC GTA TTA

B:

BW 54-x short (SEQ ID NO:3): 5′-AAT AGA TAG GTA GAC CTT T

BW 54-x (SEQ ID NO:4): 5′-pACA AGT TTT CCC TGG GCC TAT AGT GAG TCG TATTA

C:

BW 54-y (SEQ ID NO:5): 5′-pCCT GGG CCT ATA GTG AGT CGT ATT A

BW 54-y short (SEQ ID NO:6): 5′-AAT AGA TAG GTA GAC CTT TAC AAG TTT TC

C′:

BW 54-y ohne (SEQ ID NO:7): 5′-CCT GGG CCT ATA GTG AGT CGT ATT A

BW 54-y short (SEQ ID NO:8): 5′-AAT AGA TAG GTA GAC CTT TAC AAG TTT TC

D:

BW 54-x short-1 (SEQ ID NO:9): 5′-AAT AGA TAG GTA GAC CTTp

BW 54-x (SEQ ID NO:10): 5′-pACA AGT TTT CCC TGG GCC TAT AGT GAG TCG TATTA

E:

BW 54-y (SEQ ID NO:11): 5′-pCCT GGG CCT ATA GTG AGT CGT ATT A

BW 54-y short-1 (SEQ ID NO:12): 5′-AAT AGA TAG GTA GAC CTT TAC AAG TTTTp

E′:

BW 54-y ohne (SEQ ID NO:13): 5′-CCT GGG CCT ATA GTG AGT CGT ATT A

BW 54-y short-1 ohne (SEQ ID NO:14): 5′-AAT AGA TAG GTA GAC CTT TAC AAGTTT T

F:

BW 54V(SEQ ID NO:15): 5′-AAT AGA TAG GTA GAC CTT TAC AAG TTT TCC CTG GGCCp

BW 54 V-1 (SEQ ID NO:16): 5′-pATA GTG AGT CGT ATT A

F′:

BW 54 V ohne (SEQ ID NO:17): 5′-AAT AGA TAG GTA GAC CTT TAC AAG TTT TCCCTG GGC C

BW 54 V-1 ohne (SEQ ID NO:18): 5′-ATA GTG AGT CGT ATT A

G:

BW 54W (SEQ ID NO:19): 5′-AAT AGA TAG GTA GAC CTT TAp

BW 54 W-1 (SEQ ID NO:20): 5′-pAAG TTT TCC CTG GGC CTA TAG TGA GTC GTATTA

G′:

BW 54 W ohne (SEQ ID NO:21): 5′-AAT AGA TAG GTA GAC CTT TA

BW 54 W-1 ohne (SEQ ID NO:22): 5′-AAG TTT TCC CTG GGC CTA TAG TGA GTCGTA TTA

Cleavage Reactions

In a typical reaction, 2.5 L of oligomer-solution (2 M in H2O), 1 L ofborate buffer (200 mM) and 1.5 L (33 M) of an aqueous solution ofcompound 1 were irradiated for the indicated amount of time in amicrocentrifuge tube. After irradiation, all samples were evaporated todryness in vacuo. Samples that were not treated with piperidine wereimmediately dissolved in loading buffer (80% formamide v/v, 10 mM EDTA,0.1 mg/ml Xylene cyanol, 1 mg/ml brom phenol blue, 5 mM NaOH). Theremaining samples were treated with piperidine (20 L, 90° C., 30 min),evaporated to dryness and co-evaporated with H2O (20 L) twice beforedissolving them in loading buffer. The reactions were analyzed using a12% denaturating (8M urea, 25% formamide) polyacrylamide gel.Electrophoreses were performed at 2000-2500 V and were generallycomplete after 3-3.5 h. The gels were cooled to ˜4° C. and visualizedand quantified using a phosphorimaging screen (Molecular Dynamics), anda Storm 860 Scanner (Molecular Dynamics).

By annealing BW 54s with a variety of counterstrands, we built a familyof constructs (FIG. 7) inspired by a selection of sites that formedeither in the process of chemical damage of DNA or, transiently, duringenzymatic processing of DNA. Nicked DNA (constructs B and C) is involvedin DNA topological transitions, DNA repair synthesis and DNA replicationof the lagging strand. Single nucleotide gaps with 3′- and5′-phosphorylated ends (constructs D and E) can be formed by AP-lyaseactivity of DNA glycosylases, or from gaps with3′-phospholycolate-5′-phosphate ends generated as the result of 4′-Habstraction by natural antibiotics such as bleomycin or by hydroxylradicals produced by electromagnetic radiation. The structure of thelysine-enediyne conjugate 1 and the lysine-acetylene conjugate 2 areshown in FIG. 4. These compounds are equipped with potent photoactivated“warheads,” as they are known by the skilled, and satisfy Lipinski'srule of five for drug design with respect to their molecular weight(<500) and H-bonding ability (not more than 5 donors, not more than 10acceptors).

While not intending to be bound by this particular explanation of theinvention's method of operation, we envisioned that the hydrophobic“warheads” bind to the nucleic acid to occupy a hydrophobic pocketcreated by the omission of a base, whereas the mono- or bis-protonatedhydrophilic lysine residue will additionally experience strong coulombicattraction towards a terminal phosphate mono- or di-anion, as showngenerally in FIG. 8.

Four sets of target sites were constructed within BW 54. The first sitewas chosen to be opposite G₂₆ of the G₂₄G₂₅G₂₆ triad, which is a natural(but minor) cleavage site for the reaction of this conjugate withundamaged duplex DNA. The second one was opposite G₁₉ of the G₁₈G₁₉diad, which is not part of a natural binding site. The third and fourthsites were opposite G₃₄ and A₃₆, respectively. These sites were chosento ascertain whether it was possible to target a single G site or othernucleotides. We wanted to know whether the damage remains localized orpropagates to the nearby G₃₄ and G₃₉G₄₀ by the hole-hopping mechanism,which may be important when electron transfer from DNA is involved inthe “warhead” activation step. We irradiated the constructs in presenceof compound 1 or 2 (10 μM), and analyzed the resulting cleavage patternsby denaturing PAGE, followed by phosphorimaging and quantification withSAFA software (Das, R., A. Laederach, S. Pearlman, D. Herschlag and R.Altman; RNA 2005, 11, 344; a scientific paper accessible to the skilledand incorporated herein by reference in its entirety). Comparisons ofconstruct cleavages are shown in FIGS. 9-14.

All constructs showed enhanced cleavage at the respective target sites.Constructs with a “gap”, an unpaired base in the target strand,displayed the greatest effect, especially when the target was within thenatural AT-rich binding site (D, E, E′, G, G′). Gaps outside of theAT-tract were less effective (constructs F and F′) and showed migrationof damage away from the target G₁₉ base not only to the adjacent G₁₈ butalso a more remote G₇ site. Interestingly, the migration of damage wasunidirectional and no enhancement is observed at the G₂₄G₂₅G₂₆ triad. Inall cases, however, cleavage at the site targeted by the construct wasclearly enhanced. In the case of constructs without the “gap” (B, C,C′), the enhancement was also evident, albeit to a smaller degree thanin the “gapped” constructs.

In the cases where the target site was not at a (multiple) G (constructsB and D), the cleavage was distributed over an array of bases terminatedby the nearest G. Both the single G₃₄ and the G₃₉G₄₀ doublet showedincreased cleavage, but also the whole A/T area in between these twosites displayed more damage relative to the duplex DNA. Remarkably, oncethe target site is moved only two bases to G₃₄ (constructs G and G′),all the damage enhancement is localized at that site, resulting inamplification by a factor of 5-6 on what used to be a minor cleavagesite in the ds construct (FIG. 10). Localization of damage at a single Gin the presence of nearby (G)_(n) sites illustrates that dissipation ofthe damage by hole-hopping does not interfere. In order to compare thedifferent types of constructs (with/without gap, with/without phosphategroup(s)), we compared constructs C, C′, E, and E′ directly, again usingthe “undamaged” double stranded construct A as reference.

Enhancement of piperidine-induced cleavage is identical within each pairof constructs with (E and E′) and without a gap (C and C′), suggestingthat, unlike the gap, phosphate groups apparently have no influence atthe oxidative piperidine-induced DNA cleavage. On the other hand, frankphotocleavage induced by our compounds, which may occur due to directH-abstraction by radical species, is clearly enhanced both by thepresence of phosphate groups and by the hydrophobic pocket (see FIG.11).

In order to understand the role of the phosphate groups in therecognition of lysine-conjugates, we also varied the buffer conditions.The directing effect, as well as the reactivity, is much more pronouncedin borate buffer than it is in phosphate buffer. While we observe anenhancement by a factor of approximately eight in borate buffer, thedirecting efficiency of the target site is reduced by ˜50% in 20 mMphosphate buffer. These findings suggest that recognition between theammonium groups of lysine and phosphates of the DNA does contribute tothe directing effect and that this contribution decreases upon“dilution” with the external phosphate moieties. Nevertheless, even inpresence of a 4000-20000 molar excess of phosphate, the directing effectis still present, suggesting the robustness of these recognitionpatterns.

Both the robustness and the versatility of DNA damage recognition arefurther illustrated by reactivity of another lysine conjugate (accordingto formula 2) over a wider range of conditions. Differences inselectivity are minimal for experimental results for pH 6, pH 7, and pH8, and cleavage at the target site is amplified by a factor of 5-6, evenin the presence of phosphate buffer. These experiments show that theeffect is general and can be used with a variety of DNA-cleaving agentsunder a variety of conditions.

The directing effect of ss damage sites provides a new explanation forthe unusually high ratios of double strand to single strand (ds/ss)cleavage of plasmid DNA by enediyne-lysine conjugates, where statisticalevidence based on the Poisson distribution suggested a dramatic increasein the amount of ds cleavage compared to that expected from acombination of random ss cleavages. Although statistical evidence doesnot differentiate between the true ds cleavage and this alternativescenario, the possibility of directing a second attack throughrecognition of the initial ss cleavage points to a new strategy in thedesign of highly reactive ds DNA cleaving agents.

Accordingly, the present invention shows that it is possible for smallmolecules (MW<500) to recognize damage sites in one strand of doublestranded DNA, and to use this recognition to direct subsequent damage toa specified location at the counterstrand, thus converting ss cleavageto ds cleavage. While such recognition is known for enzymes and somelarge natural products, the present invention demonstrates that theunderlying working pattern can be simulated with compounds that aresmaller than enzymes by at least two orders of magnitude.

Having read the disclosure provided by the inventors, those skilled inthe art will appreciate that the invention includes a number ofembodiments, including a process of forming a double strand cleavage inDNA. This process comprises providing a reaction mixture containingdouble stranded DNA having a break in a first strand defining a targetsite in a second strand. The break defines a target site on the unbrokenstrand. The break can be created by photochemical or chemical cleavageof an intact duplex or can be created artificially by annealing a singlenucleotide strand with one or more shorter counterstrands. The methodcontinues by adding to the reaction mixture a photoreactive lysineconjugate selected from lysine-enediyne conjugate according to formulaI, a lysine-acetylene conjugate according to formula II and combinationsthereof, for a time sufficient for the lysine conjugate to bind to theDNA adjacent the target site. Finally, the method calls for irradiatingthe reaction mixture with electromagnetic radiation sufficient tophotoactivate the lysine conjugate to cleave the second strand adjacentthe target site.

Another preferred embodiment of the invention is a process for cleavingdouble stranded DNA, the process comprising reacting double stranded DNAcontaining a target site adjacent a nick or gap in a first strand with aphotoreactive lysine enediyne conjugate according to formula I or alysine acetylene conjugate according to formula II, and irradiating themixture at a wavelength effective for causing photoactivation of theconjugate to thereby cleave a second strand of the DNA adjacent thetarget site.

Yet an additional preferred embodiment of the invention is a method forsite-directed cleavage of a nucleic acid strand. This method comprisessynthesizing a complementary counterstrand bearing a terminal phosphategroup. The method then proceeds by annealing the nucleic acid strand andthe counterstrand so that the terminal phosphate on the counterstrand isopposite one or more guanosines on the nucleic acid strand. A reactionis started by mixing the annealed strands with a photoreactivephosphate-detecting ligand conjugate. Finally, the reaction mixture isirradiated at a wavelength effective for photoactivating the conjugateto cleave the nucleic acid strand at the site of the one or moreguanosines.

A further preferred method of the invention provides for site-directedcleavage of a nucleic acid strand. This method comprises synthesizing acounterstrand complementary to a predetermined region of the nucleicacid strand, the counterstrand bearing a terminal phosphate group. Themethod then calls for annealing the counterstrand with the predeterminedregion of the nucleic acid strand so that the terminal phosphate groupon the counterstrand is approximately opposite a selected target site onthe nucleic acid strand. The annealed strands are then contacted with aphotoreactive ligand conjugate effective for complexing with thecounterstrand at the terminal phosphate group. Lastly, the method endsafter irradiating the complex at a wavelength effective forphotoactivating the conjugate to cleave the nucleic acid strand at theselected target site.

Accordingly, in the drawings and specification, there have beendisclosed typical preferred embodiments of the invention, and althoughspecific terms are employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

1. A process of forming a double strand cleavage in DNA, the processcomprising: providing a reaction mixture containing double-stranded DNAhaving a break in a first strand defining a target site in a secondstrand; adding to the reaction mixture the photoreactive lysineconjugate selected from the lysine-enediyne conjugate consisting offormula 1 as shown in FIG. 4, the lysine-acetylene conjugate consistingof formula 2 as shown in FIG. 4, and combinations thereof, for a timesufficient for the lysine conjugate to bind to the DNA adjacent thetarget site; and irradiating the reaction mixture with electromagneticradiation sufficient to photoactivate the lysine conjugate to cleave thesecond strand adjacent the target site.
 2. A process for cleavingdouble-stranded DNA, the process comprising: reacting double-strandedDNA containing a target site adjacent a nick or gap in a first strandwith the photoreactive lysine-enediyne conjugate consisting of formula 1as shown in FIG. 4 or the lysine-acetylene conjugate consisting offormula 2 as shown in FIG. 4; and irradiating the reaction mixture at awavelength effective for causing photoactivation of the conjugate tothereby cleave a second strand of the DNA adjacent the target site.
 3. Amethod for site-directed cleavage of a nucleic acid strand, the methodcomprising: synthesizing a complementary counterstrand bearing aterminal phosphate group; annealing the nucleic acid strand and thecounterstrand so that the terminal phosphate on the counterstrand isopposite one or more guanosines on the nucleic acid strand; mixing theannealed strands with a photoreactive phosphate-detecting ligandconjugate; and irradiating the mixture at a wavelength effective forphotoactivating the conjugate to cleave the nucleic acid strand at thesite of the one or more guanosines.
 4. A method for site-directedcleavage of a nucleic acid strand, the method comprising: synthesizing acounterstrand complementary to a predetermined region of the nucleicacid strand, the counterstrand bearing a terminal phosphate group;annealing the counterstrand with the predetermined region of the nucleicacid strand so that the terminal phosphate group on the counterstrand isapproximately opposite a selected target site on the nucleic acidstrand; contacting the annealed strands with a photoreactive ligandconjugate effective for complexing with the counterstrand at theterminal phosphate group; and irradiating the complex at a wavelengtheffective for photoactivating the conjugate to cleave the nucleic acidstrand at the selected target site.